title: Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase.
abstract: 6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer-tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔCp, with NADP binding ΔCp closer to what is expected of crystallographic structures, while NADPH ΔCp is three times larger. The estimated dimer-tetramer association constant is 1.5·10(6)M(-1), and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.
authors.affiliation: Dept. of Biomedical and Specialty Surgical Sciences, Biochemistry Unit, Via Borsari 46, Università di Ferrara, 44121 Ferrara, Italy. Electronic address: hns@unife.it.
authors.forename: S
authors.initials: S
authors.lastname: Hanau
authors.forename: L Proietti
authors.initials: LP
authors.lastname: d'Empaire
authors.forename: I
authors.initials: I
authors.lastname: Capone
authors.forename: S
authors.initials: S
authors.lastname: Alberighi
authors.forename: R
authors.initials: R
authors.lastname: Montioli
authors.forename: F
authors.initials: F
authors.lastname: Dallocchio
mesh_terms: D000818:Animals; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D012756:Sheep; D013045:Species Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014353:Trypanosomiasis, African / Q000201:enzymology
chemical_list: D009249:NADP; D010734:Phosphogluconate Dehydrogenase
keywords: 4-phospho-erythronate; 4PE; 6-phosphogluconate; 6-phosphogluconate dehydrogenase; 6PG; 6PGDH; ASA(ap); ASA(p); DLS; Dimer–tetramer equilibrium; G6PDH; ITC; NADPH inhibition; PPP; Pentose phosphate pathway; Trypanosoma brucei; apolar solvent-accessible surface area; dynamic light scattering; glucose-6-phosphate dehydrogenase; isothermal titration calorimetry; pentose phosphate pathway; polar solvent-accessible surface area